Abstract
BackgroundSpermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to mature functional spermatozoa, being the only adult stem cells in the males that can transmit genetic information to the next generation. Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine. However, studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually.ResultsIn the present study, by lentiviral transduction of plasmids expressing the simian virus 40 (SV40) large T antigen into porcine primary SSCs, we developed two immortalized cell lines with porcine SSC attributes. The established cell lines, with the expression of porcine SSC and germ cell markers UCHL1, PLZF, THY1, VASA and DAZL, could respond to retinoic acid (RA), and could colonize the recipient mouse testis without tumor formation after transplantation. The cell lines displayed infinite proliferation potential, and have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities.ConclusionsWe have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation, thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine.
Highlights
Spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to mature functional spermatozoa, being the only adult stem cells in the males that can transmit genetic information to the generation
One day after lentiviral transduction, cells were subjected to FACS employing an antibody against PLD6
The enriched germ cells were separated to two fractions, and two distinct vectors expressing the simian virus 40 (SV40) large T antigen, namely pLOX-Ttag-iresTK and Ef1a-Large T-Ires-Puro, were introduced to two germ cell fractions respectively by the optimized lentiviral transduction
Summary
Spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to mature functional spermatozoa, being the only adult stem cells in the males that can transmit genetic information to the generation. Studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually. SSCs are capable of both self-renewal (to maintain the sufficient quantity) and differentiation (to mature functional spermatozoa), being the only adult stem cells in the males that can transmit genetic information to the generation [1]. Pigs (Sus scrofa) are a leading domestic species over the world for meat production They are increasingly exploited as an animal model in physiological and pharmacological research due to their high similarity to humans with respect to anatomy and physiology [5]. After the 2-month culture period, cell proliferation came to a standstill, along with the prevalence of differentiation and apoptosis, leading to a sharp decline in the total cell number [9], suggesting that there is large room for improvement of porcine SSC culture
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